CN100552414C - Paraffin section method for seawater fish egg - Google Patents
Paraffin section method for seawater fish egg Download PDFInfo
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- CN100552414C CN100552414C CNB2006100454298A CN200610045429A CN100552414C CN 100552414 C CN100552414 C CN 100552414C CN B2006100454298 A CNB2006100454298 A CN B2006100454298A CN 200610045429 A CN200610045429 A CN 200610045429A CN 100552414 C CN100552414 C CN 100552414C
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Abstract
A kind of paraffin section method for seawater fish egg, comprise the determining of the determining of the determining of selection, dehydration procedure, transparent process, wax penetration process of fixing agent, the improvement of colouring method, omitted in the fish-egg paraffin slicing processes unworkable egg membrane and peeled off program, simplified program, improved work efficiency.Overcome the ovum distortion, the section that exist in the seawater fish egg paraffin section discontinuous, have a problem such as cavity, obtain good section and dyeing quality, this method is simple to operation, does not need Special Equipment, medicine is easy to get, and can be used for the biological study of seawater fish early development.
Description
Technical field
The invention belongs to a kind of seawater fish egg paraffin section technology, is the method for fixing agent, dehydration procedure, transparent process, saturating wax method and colouring method being carried out major tuneup on the basis of existing paraffin section technology.
Background technology
Before the present invention makes, paraffin section is because simple to operate, cheap characteristics, it is traditional biological study technology always, paraffin section comprises: steps such as fixing, dehydration, transparent, saturating wax, embedding, section, paster, dewaxing, dyeing, transparent and mounting have all obtained using widely in biological many fields.But because seawater fish egg has special egg membrane structure, the effect of its paraffin section is unsatisfactory always, for the fresh water fish-egg, has adopted both at home and abroad to divest the method for carrying out paraffin section behind the egg membrane by classic method, and effect obviously improves.But, divest the egg membrane operating difficulties and be easy to cause ovum to break because all cracks of the ovum of seawater fish ovum are less; The ovum that does not divest egg membrane is when cutting into slices, because often there are problems such as ovum distortion, section fragmentation in the barrier effect of egg membrane; Aspect fixing agent, adopt light microscopic sample fixing agent commonly used, undesirable for the fixed effect of seawater fish egg sample, it is imperfect to cause cutting into slices; In dehydration procedure, because fish-egg has egg membrane, influence the inside and outside material exchange of egg membrane, conventional dewatering can cause the ovum distortion owing to dehydration is too fast; In transparent process, the fish-egg after the dehydration is out of shape rapidly after entering dimethylbenzene, and irrecoverable; In wax penetration process, because the paraffin molecule amount is bigger, egg membrane is stronger to its barrier effect, causes wax incomplete; Never have effect paraffin section method for seawater fish egg preferably at present, the electron microscopic observation method that some researcher can only adopt expense, equipment to have relatively high expectations has limited the biological research of seawater fish early development.
Summary of the invention
The purpose of this invention is to provide and a kind ofly can improve seawater fish egg paraffin section method for quality, enforcement by this method, can omit the time-consuming egg membrane step that divests, simplify and improve the effect of seawater fish egg paraffin section, the ovum in the solution seawater fish egg paraffin section process is out of shape, wax disk(-sc) is discontinuous, empty problem is arranged.
The present invention finishes by following operative technique:
Paraffin section comprises: fixing, dehydration, transparent, saturating wax, embedding, section, paster, dewaxing, dyeing, transparent and mounting, the present invention is in seawater fish egg paraffin section process, fixing agent, dehydration procedure, transparent process, saturating wax method, embedding program and colouring method are improved, promptly carried out the determining of the determining of the determining of selection, dehydration procedure, transparent process, wax penetration process of fixing agent, the improvement of colouring method.
The selection of fixing agent: adopt the fixing glutaraldehyde commonly used of electron microscopic sample, the concentration of glutaraldehyde is 1.5%, and the seawater that adopts the absorbent cotton filtration is as solvent.Change immobile liquid 3～4 times in the fixation procedure.
Determining of dehydration procedure: sample adopts ethanol dehydration, adopts: 85% ethanol 50min, 95% ethanol 45min, 95% ethanol 45min, 100% ethanol 35min, 100% ethanol 35min.85% to 95% ethanol is taked 10% concentration gradient, and 95% to 100% ethanol is taked 5% concentration gradient, and 2 dehydration procedures of every gradient are located the sample embedding with 100% agar before the sample dehydration.
Determining of transparent process: take 1: 1 dimethylbenzene: the ethanol transition method.Concrete transparent process is as follows: 1: 1 dimethylbenzene: 100% ethanol 15s, dimethylbenzene 10s, dimethylbenzene 10s.
Determining and embedding of wax penetration process: it is 42～47 ℃ soft wax that saturating wax adopts fusing point, and wax penetration process is as follows: the saturating 3 hours wax time, during change wax once, i.e. paraffin 1h30min, paraffin 1h30min uses the carton embedding behind the saturating wax.Adopt common biological microtome section, slice thickness 5 μ m; Open up sheet with water bath with thermostatic control, the animal glue paster.
The improvement of colouring method: dyeing has adopted the eosin haematoxylin to redye, and the haematoxylin formula for dye liquor is as follows: hot distilled water 2000ml, haematoxylin 2g, sodium iodate 0.4g, potassium alum 150g, citric acid 2g, water and chloral 100g; Eosin formula for dye liquor: 1% eosin W or W S aqueous solution 100ml, 98% ethanol 780ml, glacial acetic acid 4ml.
The present invention and prior art contrast are characterized in:
1, the present invention has omitted in the fish-egg paraffin slicing processes unworkable egg membrane and has peeled off program, has simplified program, has improved work efficiency.
2, the present invention overcome the ovum distortion, the section that exist in the seawater fish egg paraffin section discontinuous, have problem such as cavity, slice thickness can reach 5 μ m.
3, the inventive method is simple to operate, does not need Special Equipment, and medicine is easy to get, and is convenient to promote the use of.
Description of drawings:
The spindle in the ovum cortex in the meiosis is shown in Fig. 1, the section of turbot ovum.
1-ovum cortex, 2-spindle
The spindle in the ovum in the spilting of an egg is shown in Fig. 2, the section of turbot ovum.
The 2-spindle
Embodiment:
Be described in detail technical method of the present invention in conjunction with the accompanying drawings below by embodiment:
Paraffin section comprises: fixing, dehydration, transparent, saturating wax, embedding, section, dewaxing, dyeing, transparent and mounting.The present invention is in seawater fish egg paraffin section process, fixing agent, dehydration procedure, transparent process, saturating wax method, embedding program and colouring method are improved, promptly carried out the determining of the determining of the determining of selection, dehydration procedure, transparent process, wax penetration process of fixing agent, the improvement of colouring method.
The selection of fixing agent: light microscopic sample fixing agent commonly used, unsatisfactory for the fixed effect of seawater fish egg sample, the normal section situation such as break that occurs in slicing processes.We adopt the fixing glutaraldehyde commonly used of electron microscopic sample, have obtained good fixed effect, and section is continuous, and the situation of breaking significantly reduces.The concentration of glutaraldehyde is 1.5%, and in order to guarantee the consistance of immobile liquid and fish-egg osmotic pressure, the seawater that adopts the absorbent cotton filtration is as solvent.In order to guarantee the concentration of glutaraldehyde immobile liquid, change immobile liquid 3～4 times in the fixation procedure.
Dehydration procedure: because fish-egg has egg membrane, influence the inside and outside material exchange of egg membrane, conventional dewatering can be because the too fast ovum distortion that causes of dewatering, through constantly revising dehydration procedure, the dehydration procedure that we adopt is as follows: 85% ethanol 50min, 95% ethanol 45min, 95% ethanol 45min, 100% ethanol 35min, 100% ethanol 35min; 85% to 95% ethanol is taked 10% concentration gradient, and 95% to 100% ethanol is taked 5% concentration gradient, and the dehydration procedure that every gradient is 2 times has obtained dehydrating effect preferably, and the ovum dehydration does not have contraction, distortion situation fully substantially.With 100% agar the sample embedding is located before the sample dehydration in addition, can make things convenient for later operation like this.
Transparent process: find that in experimentation the fish-egg after the dehydration is out of shape rapidly after entering dimethylbenzene, and irrecoverable, for this reason, we have taked 1: 1 dimethylbenzene: the method for ethanol transition has solved this problem well.Concrete transparent process is as follows: 1: 1 dimethylbenzene: 100% ethanol 15s, dimethylbenzene 10s, dimethylbenzene 10s.
Saturating wax, embedding and section program: because the paraffin molecule amount is bigger, egg membrane is very strong to its barrier effect, therefore, we have adopted the long saturating wax time, cause the sample embrittlement in order to prevent the wax overlong time, and we adopt the lower paraffin of fusing point again, like this, obtained good effect, saturating wax is complete, and it is complete to cut into slices.It is 42～47 ℃ soft wax that saturating wax adopts fusing point, and wax penetration process is as follows: the saturating 3 hours wax time, during change wax once, i.e. paraffin 1h30min, paraffin 1h30min uses the carton embedding behind the saturating wax.Adopt common biological microtome section, slice thickness 5 μ m; Open up sheet with water bath with thermostatic control, the animal glue paster.
Dewaxing, dyeing and mounting method: the dimethylbenzene dewaxing is taked in dewaxing, specific procedure is as follows: dimethylbenzene 5min, and dimethylbenzene 5min carries out rehydration then, specific procedure is as follows: 100% ethanol 2min, 100% ethanol 2min, 95% ethanol 2min, 95% ethanol 2min, 80% ethanol 2min, 80% ethanol 2min, 50% ethanol 2min, distilled water 2min; Section after rehydration eosin haematoxylin complex staining.There is document to show, the glutaraldehyde immobile liquid can influence the dyeing situation of section, causes dyeing shallow, and color separation is not obvious, we have carried out the dyeing observation to the section of 1.5% glutaraldehyde seawater immobile liquid fixed sample for this reason, dyeing has adopted the eosin haematoxylin to redye, and the haematoxylin formula for dye liquor is as follows: hot distilled water 2000ml, haematoxylin 2g, sodium iodate 0.4g, potassium alum 150g, citric acid 2g, water and chloral 100g; Eosin formula for dye liquor: 1% eosin W or W S aqueous solution 100ml, 98% ethanol 780ml, glacial acetic acid 4ml.Specific procedure is as follows: haematoxylin 3min50s, tap water flushing 4min, eosin 2min30s; The result shows that the glutaraldehyde immobile liquid is to not significantly influence of dyeing, and section statining is respond well, and dye level is suitable, and color separation is obvious.Section dehydration of alcohol after the dyeing, dimethylbenzene is transparent, and specific procedure is as follows: 95% ethanol 2min, 95% ethanol 2min, 100% ethanol 2min, 100% ethanol 2min, dimethylbenzene 2min30s, dimethylbenzene 2min30s, dimethylbenzene 2min30s uses the canada balsam mounting then.
Adopt said method, we have carried out practical application in Inst of Huanghai Sea Marine Products, Chinese Academy of Aquatic Product Science, more than ten batches of ovum to marine fish such as turbot, lefteye flounders has carried out paraffin section altogether, the result shows, adopt method of the present invention can obtain good section and dyeing quality, it is continuous, complete to cut into slices, and fine structure such as spindle is high-visible in the section, and concrete effect is seen (Fig. 1,2).
Claims (1)
1, a kind of paraffin section method for seawater fish egg, its running program is: fixing, dehydration, transparent, saturating wax, embedding, section, dewaxing and dyeing, it is characterized in that in seawater fish egg paraffin section process, fixing agent, dehydration procedure, transparent process, saturating wax method and colouring method are improved, have promptly carried out the determining of the determining of the determining of selection, dehydration procedure, transparent process, wax penetration process of fixing agent, the improvement of colouring method:
The selection of fixing agent: adopt the fixing glutaraldehyde commonly used of electron microscopic sample, the concentration of glutaraldehyde is 1.5%, and the seawater that adopts the absorbent cotton filtration is as solvent.Change immobile liquid 3～4 times in the fixation procedure;
Determining of dehydration procedure: sample adopts ethanol dehydration, adopts: 85% ethanol 50min, 95% ethanol 45min, 95% ethanol 45min, 100% ethanol 35min, 100% ethanol 35min; 85% to 95% ethanol is taked 10% concentration gradient, and 95% to 100% ethanol is taked 5% concentration gradient, and 2 dehydration procedures of every gradient are located the sample embedding with 100% agar before the sample dehydration;
Determining of transparent process: take 1: 1 dimethylbenzene: the ethanol transition method; Concrete transparent process is as follows: 1: 1 dimethylbenzene: 100% ethanol 15s, dimethylbenzene 10s, dimethylbenzene 10s;
Determining and embedding of wax penetration process: it is 42～47 ℃ soft wax that saturating wax adopts fusing point, and wax penetration process is as follows: the saturating 3 hours wax time, during change wax once, i.e. paraffin 1h30min, paraffin 1h30min uses the carton embedding behind the saturating wax; Adopt common biological microtome section, slice thickness 5 μ m; Open up sheet with water bath with thermostatic control, the animal glue paster;
The improvement of colouring method: dyeing has adopted the eosin haematoxylin to redye, and the haematoxylin formula for dye liquor is as follows: hot distilled water 2000ml, haematoxylin 2g, sodium iodate 0.4g, potassium alum 150g, citric acid 2g, water and chloral 100g; Eosin formula for dye liquor: 1% eosin W or W S aqueous solution 100ml, 98% ethanol 780ml, glacial acetic acid 4ml.
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| CNB2006100454298A CN100552414C (en) | 2006-07-10 | 2006-07-10 | Paraffin section method for seawater fish egg |
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| CN100552414C true CN100552414C (en) | 2009-10-21 |
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Families Citing this family (16)
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| CN101545876B (en) * | 2009-04-29 | 2011-02-16 | 中国海洋大学 | Method for observing collagenous fibre of fresh and alive trepang with electron microscope |
| CN102062709B (en) * | 2010-12-13 | 2012-08-29 | 苏州大学 | Method for quickly slicing fish tissues |
| CN102147337B (en) * | 2011-01-13 | 2012-11-14 | 中国水产科学研究院东海水产研究所 | Paraffin sectioning method for ocean shellfish D-shaped larva |
| CN102116711B (en) * | 2011-01-31 | 2012-11-21 | 山东东方海洋科技股份有限公司 | Manufacturing method of paraffin sections of zostera marina embryo |
| CN102221484A (en) * | 2011-04-21 | 2011-10-19 | 中国水产科学研究院东海水产研究所 | Gonad slicing method for pomfret larvae juvenile fish |
| EP2546628A1 (en) * | 2011-07-13 | 2013-01-16 | Koninklijke Philips Electronics N.V. | Filter support with a phase-changing medium |
| CN102279116A (en) * | 2011-07-14 | 2011-12-14 | 北京大学第三医院 | Method for preparing membrane-type sample slice |
| CN102313660A (en) * | 2011-10-09 | 2012-01-11 | 中国科学院南海海洋研究所 | Manufacturing method of paraffin section of hermatypic coral oocyte |
| CN102589949B (en) * | 2012-02-29 | 2013-07-24 | 中国水产科学研究院黑龙江水产研究所 | Dehydration tool used in preparation of fish ovum tissue slice |
| CN102607930A (en) * | 2012-03-13 | 2012-07-25 | 中国水产科学研究院黑龙江水产研究所 | Fish germ cell orientated embedding technology |
| CN103954484B (en) * | 2014-04-02 | 2016-08-24 | 浙江万里学院 | A kind of manufacture method of Trionyx sinensis (Wiegmann) gonad paraffin section in early days |
| CN103940648B (en) * | 2014-04-04 | 2016-02-24 | 山西农业大学 | The preparation method of fish gill tissue paraffin section |
| CN105241686B (en) * | 2015-08-10 | 2018-04-13 | 河南科技大学 | The preparation method of Hynobiidae animal retina microscopic tissue sections |
| CN105571916B (en) * | 2015-12-24 | 2018-08-17 | 四川省农业科学院水产研究所 | A kind of tissue section method of acipenser dabryanus fertilized eggs |
| CN106053187A (en) * | 2016-05-20 | 2016-10-26 | 北京九州柏林生物科技有限公司 | Liquid set for dyeing biological tissue sample slices |
| CN111487096A (en) * | 2019-01-25 | 2020-08-04 | 南京理工大学 | Method for analyzing distribution and damage degree of micro-plastic by using zebra fish |
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| WO2003029783A1 (en) * | 2001-09-28 | 2003-04-10 | Jurgen Olert | Tissue fixative composition |
| CN1749730A (en) * | 2004-09-17 | 2006-03-22 | 中国科学院海洋研究所 | A kind of tissue section method of seawater left-eyed flounder zygote |
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Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2003029783A1 (en) * | 2001-09-28 | 2003-04-10 | Jurgen Olert | Tissue fixative composition |
| CN1749730A (en) * | 2004-09-17 | 2006-03-22 | 中国科学院海洋研究所 | A kind of tissue section method of seawater left-eyed flounder zygote |
Non-Patent Citations (1)
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